1. Field of the Invention
The field of this invention is transgenic flies.
2. Background of the Invention
Adult onset neurodegenerative diseases are among some of the most devastating diseases currently afflicting mankind, at least in the developed nations of the world. Such diseases xe2x80x9care characterized by onset in adult life, chronic progressive course, distinct clinical phenotypes, specific cellular abnormalities involving subsets of neurons, and eventually fatal outcomes.xe2x80x9d Price et al., Science (Nov. 6, 1998) 282: 1079-1083. Examples of neurodegenerative diseases include: Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, Huntington""s disease, and the like. Currently, treatment of such diseases is limited to disease management and there are no cures. As such, there is much ongoing research that is aimed at the identification and development of new therapeutic agents which can at least slow, if not reverse, the progression of neurodegenerative diseases.
Critical steps in the identification and development of new therapeutic agents are: (a) the generation of candidate agents; and (b) screening of the candidate agents for efficacy (and safety). With the advent of combinatorial chemistry protocols, large numbers of potential compounds, known as libraries, can be rapidly generated. Such libraries serve as a collection of potential therapeutic agents. Following generation of a library of potential therapeutic agents, the library must be screened to identity the promising candidates.
For screening purposes, a number of in vitro high throughput screening protocols have been developed. However, these in vitro screening assays must be followed by in vivo screening assays. Since it is undesirable to immediately screen compounds that show promise from in vitro assays in humans, an important step in the identification of therapeutic agents for such neurodegenerative diseases is the screening of potential therapeutic compounds in non-human animal models. As such, non-human animal models of neurodegenerative diseases play an important role in the discovery of therapeutic agents for such diseases.
One type of non-human animal model that can be used for screening purposes to identify therapeutic agents for use in treating neurodegenerative diseases is a non-human mammalian model, e.g. primates, mice, etc. However, primates are expensive, difficult to use, and require a significant period of time prior to developing adult onset neurodegenerative symptoms. These factors make the use of primates as adult onset neurodegenerative animal models prohibitive. Transgenic mice suffer from analogous disadvantages, i.e. expense, slow reproduction time, and generation of small numbers of offspring.
As such, there is a need for additional animal models of adult onset neurodegenerative diseases. Of particular interest would be the development of an animal model having a relatively short life span and a rapid reproduction cycle characterized by the production of large numbers of offspring. Preferably, such an animal model should also be relatively simple and economic to maintain.
Relevant Literature
Patents of interest include: U.S. Pat. No. 4,670,388. Methods of preparing transgenic Drosophila melanogaster are disclosed in: Spradling, A. C., and Rubin, G. M. (1982). Science 218, 341-347; Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379. See also, Spradling A C, P Element Mediated Transformation in Drosophila: A Practical Approach (ed. D. D. Roberts, IRL Press, Oxford) (1986) pp 175-179. Articles disclosing drosophila that exhibit neurodegenerative phenotypes include: Buchanan and Benzer, Neuron (May 1993) 10: 839-850; Jackson et al, Neuron (September 1998) 21: 633-642; Kretzchmar et al., J. Neuroscience (Oct. 1, 1997) 17: 7425-7432; Marfany et al., J. Neurogenetics (1998) 12: 41-54; Min and Benzer, Current Biology (1997) 7:885-888; Rogina et al., Proc. Nat""l Acad. Sci. USA (June 1997) 94: 6303-6306; and Warrick et al., Cell (Jun. 12, 1998) 93: 939-949.
Articles describing non-human animal models for neurodegenerative diseases include: Price et al., Science (Nov. 6, 1998) 282:1079-1083.
Transgenic non-mammalian animals, e.g. flies, that exhibit an adult onset neurodegenerative phenotype, as well as methods for preparing the same, are provided. The subject transgenic animals are characterized in that they have a transgene stably integrated into their genome the expression of which in embryonic neuroblasts results in the adult onset neurodegenerative phenotype. In preferred embodiments, the transgene is a myb gene. Also provided are methods of screening compounds for activity with respect to adult onset neurodegeneration, particularly compounds having therapeutic activity with respect to adult onset neurodegenerative disease conditions.
Non-mammalian transgenic animals, particularly insects, e.g. flies, that exhibit adult onset neurodegeneration, as well as methods for their preparation, are provided. The subject transgenic animals are further characterized in that they comprise a stably integrated transgene which is expressed in embryonic neuroblasts, where the expressed product results in the observed adult onset neurodegenerative phenotype. In preferred embodiments, the transgene is a myb gene. Also provided are methods of using the subject non-mammalian transgenic animals to screen for compounds having activity with respect to adult onset neurodegeneration, particularly compounds that are therapeutic for neurodegenerative diseases. In further describing the subject invention, the transgenic animals and methods for their production will be detailed first, followed by a discussion of the screening methods of the subject invention.
Animal Models
In the broadest sense, the invention provides non-mammalian animal models that exhibit an adult onset neurodegenerative phenotype, where the phenotype results from a non-adult expression event. In other words, the phenotype of the animal models of the subject invention results from an expression event that occurs in the non-adult animal. The phenotype may result from a variety of different non-adult expression events, where the expression event may be the expression of a mutated gene, the failure of expression of a normally expressed gene, the expression of a transgene, and the like.
In many preferred embodiments, the subject invention provides non-mammalian transgenic animals (i.e. multi-cellular non-plant organisms) that have an adult onset neurodegenerative phenotype. In other words, the subject transgenic animals exhibit one or more phenotypic traits that characterize neurodegenerative disorders and occur in the adult organism (e.g. during the adult stage of the organism""s life), where such traits include: impaired motor skills, impaired cognitive skills, reduced appetite, etc. As such, the subject animals are non-mammalian animal models for neurodegenerative disorders, particularly adult onset neurodegenerative disorders, such as: Parkinson""s Disease, Alzheimer""s Disease, Huntington""s Disease, stroke, and the like. The term transgenic animal is used broadly herein to refer to a non-plant multicellular organism at any stage of development, e.g. adult, fertilized eggs, embryos, larva, etc. Thus, a particular multicellular organism is a transgenic animal according to the subject invention no matter which stage of development it is at, so long as the animal exhibits the requisite adult onset neurodegenerative phenotypic characteristics that results from the correct spatial and temporal expression of the transgene stably integrated into its genome, as described in greater detail infra.
The transgenic animals of the subject invention are non-mammalian transgenic animals. Of particular interest are invertebrate transgenic animals, particularly members of the phylum arthropoda, and more particularly members of the class insecta. Of particular interest in many embodiments are transgenic flies. In many preferred embodiments, the transgenic flies are members of the family Drosophilidae, where the transgenic animal is often a Drosophila melanogaster. The subject invention is now further described in terms of transgenic flies.
Transgenic flies of the subject invention, e.g. Drosophila melanogaster, exhibit the following phenotypic characteristics: normal wild type phenotype through the initial hours of adult life, where by initial hours is meant at least the first 1 hour, usually at least the first 3 hours and more usually at least the first 5 hours, where in many embodiments the flies may exhibit a normal phenotype through the first 10 to 15 hours of adult life or longer, but in many instances the normal phenotype will cease by about 12 to 13 hours of adult life. Following the initial hours of adult life, the flies exhibit one or more phenotypic characteristic of neurodegeneration, including: progressive loss of neuromuscular control, e.g. of the wings; progressive degeneration of general coordination; progressive degenerative of locomotion; and progressive degeneration of appetite. The subject transgenic flies are further characterized in that death occurs at 4 to 6, usually 4 to 5 days of adult life.
A critical feature of the subject transgenic animals is that the animals harbor a stably integrated transgene that is spatially and temporally expressed in a manner sufficient to result in the desired adult onset neurodegenerative phenotype. The term xe2x80x9ctransgenexe2x80x9d is used herein to describe genetic material which has been or is about to be artificially inserted into the genome of a cell. With regards to spatial expression of the transgene, expression is generally limited to neuronal cells, specifically neuroblasts. With regards to temporal expression, the transgene is required to be expressed in the embryo. In other words, the transgenic expression occurs transiently in the embryonic neuroblasts.
The transgene encodes a product that, when expressed in embryonic neuroblasts, gives rise to the adult onset neurodegenerative phenotype. Generally, the transgene encodes a transcription factor or mimetic thereof having the desired result. Suitable transgenes are those that encode products which activate transcription and can bind to a Myb DNA binding sequence present in the genome of the transgenic animal. Of interest in many embodiments is a myb gene, where the myb gene may be a naturally occurring myb gene or a gene that encodes a product having the desired structural and functional properties, e.g. DNA binding, of the product expressed by a naturally occurring myb gene. Thus, the transgene may comprise a nucleic acid having the sequence of a naturally occurring myb gene or a sequence that includes only a myb domain that governs sequence specific DNA binding (i.e. a myb DNA binding domain) and a transcriptional activation domain. As such, the transgene at least includes a myb DNA binding domain or a sequence that is substantially similar to a naturally occurring myb DNA domain to provide for the requisite DNA binding activity, where substantially similar means a nucleic acid sequence having a sequence identity with a naturally occurring myb DNA binding domain of at least about 50%, usually at least about 60% and more usually at least about 65%, where sequence identity is determined using the BLAST program at default settings. In many embodiments, the transgene comprises a naturally occurring myb gene or a sequence substantially similar thereto. Of particular interest as the transgene is a non-Drosophila melanogaster myb gene, preferably a chicken or viral myb gene. Thus, preferred transgenes found in the subject animals are viral or chicken myb genes that encode a product that activates transcription and binds to DNA at a Myb binding sequence. Chicken myb genes of interest include: A-myb and C-myb; while viral myb genes of interest include: v-myb-1151; v-myb-ds7, v-myb-1120 and v-myb-859 (Ganter, B. and J. S. Lipsick (1999) Adv. Cancer Res. (In Press); Ma, X. and B. Calabretta (1994) Cancer Res. 54: 6512-6516; J. S. Lipsick (1996) Oncogene. 13: 223-235; Katzen, A. L., et al., (1998). Genes Dev. 12: 831-843; Garrido, C., et al., (1992) J. Vir. 66: 6773-6776; Fu, S. L. and J. S. Lipsick (1997) Cell Growth Diff. 8: 35-45; Grasser, F. A., et al., (1992) Oncogene 7: 1005-1009; Latham, K. E., et al., (1996) Oncogene 13: 1161-1168; Lane, T., et al., (1990) Mol. Cell. Biol. 10: 2591-2598; Chen, R. H., Fields, S. and J. S. Lipsick (1995) Oncogene 11: 1771-1779; Chen, R. H. and J. S. Lipsick (1993) Mol. Cell. Biol. 13: 4423-4431; Fu, S. L. and J. S. Lipsick (1996) J. Vir. 70: 5600-5610).
As expression of the transgene is targeted to occur in a non-adult stage of the animal, the transgene is stably integrated into the genome of the animal in a manner such that its expression is controlled both spatially and temporally to the desired cell type and the correct developmental stage, i.e. to expression in embryonic neuroblasts. Specifically, the subject transgene is stably integrated into the genome of the animal under the control of a promoter that provides for expression in embryonic neuroblasts. The transgene may be under the control of any convenient promoter that provides for this requisite spatial and temporal expression pattern, where the promoter may be endogenous or exogenous, but will generally be endogenous. A suitable promoter is the promoter located in the Drosophila melanogaster genome at position 86E1-3.
The transgene may be integrated into the fly genome in a manner that provides for direct or indirect expression activation by the promoter, i.e. in a manner that provides for either cis or trans activation of gene expression by the promoter. In other words, expression of the transgene may be mediated directly by the promoter, or through one or more transactivating agents. Where the transgene is under direct control of the promoter, i.e. the promoter regulates expression of the transgene in a cis fashion, the transgene is stably integrated into the genome of the fly at a site sufficiently proximal to the promoter and in frame with the promoter such that cis regulation by the promoter occurs.
In yet other embodiments where expression of the transgene is indirectly mediated by the endogenous promoter, the promoter controls expression of the transgene through one or more transactivating agents, usually one transactivating agent, i.e. an agent whose expression is directly controlled by the promoter and which binds to the region of the transgene in a manner sufficient to turn on expression of the transgene. Any convenient transactivator may be employed, where the GAL4 transactivator system is particularly preferred in many embodiments of the subject invention.
In these preferred embodiments of the subject invention in which the transgenic fly comprises the GAL4 targeted expression system, a GAL4 encoding sequence is stably integrated into the genome of the animal in a manner such that it is operatively linked to the endogenous promoter that provides for expression solely in embryonic neuroblasts. Flies of line 31-1 (subsequently referred to as 1822), as disclosed in Brand and Perrimon, Development (1993) 118: 401-415 express GAL4 in this manner, and are known to those of skill in the art. The transgene is stably integrated into a different location of the genome, generally a random location in the genome, where the transgene is operatively linked to an upstream activator sequence, i.e. UAS sequence, to which GAL4 binds and turns on expression of the transgene. Transgenic flies having a UAS: GAL4 transactivation system are known to those of skill in the art and are described in Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379.
Methods of Producing the Subject Transgenic Flies
The subject transgenic flies can be prepared using any convenient protocol that provides for stable integration of the transgene into the fly genome in a manner sufficient to provide for the requisite spatial and temporal expression of the transgene, i.e. in embryonic neuroblasts. A number of different strategies can be employed to obtain the integration of the transgene with the requisite expression pattern. Generally, methods of producing the subject transgenic flies involve stable integration of the transgene into the fly genome. Stable integration is achieved by first introducing the transgene into a cell or cells of the fly, e.g. a fly embryo. The transgene is generally present on a suitable vector, such as a plasmid. Transgene introduction may be accomplished using any convenient protocol, where suitable protocols include: electroporation, microinjection, vesicle delivery, e.g. liposome delivery vehicles, and the like. Following introduction of the transgene into the cell(s), the transgene is stably integrated into the genome of the cell. Stable integration may be either site specific or random, but is generally random.
Where integration is random, the transgene is typically integrated with the use of transposase. In such embodiments, the transgene is introduced into the cell(s) within a vector that includes the requisite P element, terminal 31 base pair inverted repeats. Where the cell into which the transgene is to be integrated does not comprise an endogenous transposase, a vector encoding a transposase is also introduced into the cell, e.g. a helper plasmid comprising a transposase gene, such as pTURBO (as disclosed in Steller and Pirrotta, xe2x80x9cP Transposons Controlled by the Heat Shock Promoter,xe2x80x9d Mol. Cell. Biol. (1986) 6:1640-1649). Methods of random integration of transgenes into the genome of a target Drosophila melanogaster cell(s) are disclosed in U.S. Pat. No. 4,670,388, the disclosure of which is herein incorporated by reference.
In those embodiments in which the transgene is stably integrated in a random fashion into the fly genome, means are also provided for selectively expressing the transgene at the appropriate time during development of the fly. In other words, means are provided for obtaining targeted expression of the transgene. To obtain the desired targeted expression of the randomly integrated transgene, integration of particular promoter upstream of the transgene, as a single unit in the P element vector may be employed. Alternatively, a transactivator that mediates expression of the transgene may be employed. Of particular interest is the GAL4 system described in Brand and Perrimon, supra.
In this particular embodiment, the subject transgenic flies are produced by: (1) generating two separate lines of transgenic flies: (a) a first line that expresses GAL4 in embryonic neuroblasts, e.g. under the control the endogenous fly promoter located at position 86E1-3, described supra; and (b) a second line in which the transgene is stably integrated into the cell genome and is fused to a UAS domain; (2) crossing the two lines; and (3) screening the progeny for the desired phenotype, i.e. adult onset neurodegeneration. Each of the above steps are well known to those of skill in the art. See e.g. Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379. See also the Experimental Section, infra.
The above strategy is employed to obtain fertilized eggs that comprise the transgene stably integrated into the genome in a manner such that it is expressed in the correct spatial and temporal manner so that the eggs give rise to adult flies exhibiting the desired adult onset neurodegenerative phenotype. Generally, the fertilized eggs are grown at a temperature that results in the adult onset neurodegenerative phenotype, where the temperature ranges from about 24 to 30xc2x0 C., usually from about 27 to 29xc2x0 C. and more usually at about 28xc2x0 C.
Utility
The subject flies find use in a variety.of applications, including: as tools for use in the elucidation of genetic mechanisms involved in neurodegenerative disorders; as a screening tool that identifies therapeutic compounds for use in the treatment of neurodegenerative conditions (i.e. as animal models for human AON disease conditions); and as tools for use in the identification of adult onset neurodegeneration gene targets, i.e. genes whose expression can be modulated, e.g. enhanced or disrupted, in order to alleviate adult onset neurodegeneration. The subject transgenic flies find particular use in screening methods designed to identify therapeutic agents for use in the treatment of neurodegenerative diseases.
Screening Methods
As mentioned above, the subject transgenic flies find particular utility in screening assays designed to identify therapeutic compounds for neurodegenerative conditions, particularly adult onset neurodegenerative conditions. Through use of the subject transgenic flies (or cells derived therefrom depending on the particular screening assay), one can identify compounds that have activity with respect to an adult onset neurodegenerative disease. Compounds have activity with respect to an adult onset neurodegenerative disease if they modulate or have an effect on at least one parameter or symptom of the disease, such as loss of appetite, loss of motor coordination, etc., where the modulatory activity may be to reduce or enhance the magnitude of the symptom. Thus, the screening methods of subject invention can be used to identify compounds that modulate the progression of neurodegenerative disease, e.g. by binding to, modulating, enhancing or repressing the activity of a protein or peptide involved in the progression of the neurodegenerative disease, and/or compounds that ameliorate, alleviate or even remove the phenotypic symptoms of the disease, where such activity may or may not be the result of activity with respect to the underlying mechanism of the disease. Screening to determine drugs that lack effect on the neurodegeneration condition is also of interest. Assays of the invention make it possible to identify compounds which ultimately: (1) have a positive affect with respect to a neurodegenerative disease condition and as such are therapeutics, e.g. agents which arrest or reverse the neurodegeneration or ameliorate or alleviate the symptoms of such a condition; or (2) have an adverse affect with respect to the neurodegeneration disease and as such should be avoided as therapeutic agents.
In the screening methods of the subject invention, a quantity of a candidate agent is generally orally administered to the fly. Following oral administration, the affect of the candidate agent on the adult onset neurodegenerative phenotype of the fly is determined, typically by comparison with a control (i.e. a transgenic fly to which the candidate agent has not been administered). The affect of the candidate agent is determined by determining whether one or more of the phenotypic characteristics of adult onset neurodegeneration are exacerbated or ameliorated in the test fly as compared to the control fly, where characteristics that are monitored include neuromuscular control of wings, general coordination, locomotion, appetite, and the like. The candidate agent is generally orally administered to the fly by mixing the agent into the fly nutrient medium and placing the medium in the presence of the fly, (either the larva or adult fly, usually the adult fly) such that the fly feeds on the medium. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations of candidate agent. Typically, one of these concentrations serves as a negative control, i.e. no compound. In a preferred embodiment, a high throughput screening protocol is employed, in which a large number of candidate agents are tested in parallel using a large number of flies. By xe2x80x9clarge numberxe2x80x9d is meant a plurality, where plurality means at least 50, usually at least 100, and more usually at least 1000, where the number of may be 10,000 or 50,000 or more, but in many instances will not exceed 5000.
The subject methods find use in the screening of a variety of different potentially therapeutic candidate agents. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. New potential therapeutic agents may also be created using methods such as rational drug design or computer modelling.
Screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design. Candidate agents having therapeutic activity with respect to adult onset neurodegeneration can be identified based on their ability to at least ameliorate, if not completely alleviate or remove, one or more of the neurodegenerative phenotypes of the adult transgenic fly of the subject invention, such as loss of wing neuromuscular control, and the like, as described above.
The above screening methods may be part of a multi-step screening process of evaluating candidate therapeutic agents for their efficacy (and safety) in the treatment of neurodegenerative diseases in mammalian hosts, e.g. humans. In multi-step screening processes of the subject invention, a candidate compound or library of compounds is subjected to screening in a second in vivo model, e.g. a mouse model, following screening in the subject transgenic animal model. Following the initial screening in the non-mammalian transgenic animals of the subject invention, the positive compounds are then screened in non-human mammalian animal models, including transgenic non-human mammalian animal models. Transgenic mouse models of neurodegenerative diseases and methods for their use in screening assays are described in: U.S. Pat. Nos.: 5,777,194; 5,767,337; 5,720,936; 5,672,805; 5,612,486; 5,602,309; and 5,387,742, the disclosures of which are herein incorporated by reference. In addition, a pre in vivo screening step may be employed, in which the compound is first subjected to an in vitro screening assay for its potential as a therapeutic agent in the treatment of neurodegenerative conditions. Any convenient in vitro screening assay may be employed, where a variety of suitable in vitro screening assays are known to those of skill in the art.
Identification of Gene Targets
In addition to their use as animal models for screening candidate therapeutic agents, the subject transgenic flies also find use in the identification of adult onset neurodegeneration gene targets, i.e. genes whose expression can be beneficially modulated to treat adult onset neurodegeneration. Gene based therapies can be identified by doing traditional enhancer/suppressor analyses in the subject transgenic flies. In these analyses, genes in the subject transgenic flies are mutated to identify ones that either exacerbate or alleviate the adult onset neurodegeneration mutant phenotype. Methods of mutating genes and carrying out enhancer/suppressor analyses are well known to those of skill in the art (Hays, T S et al., Molecular and Cellular Biology (March 1989) 9(3):875-84; Deuring, R; Robertson, B; Prout, M; and Fuller, M T. Mol. Cell. Biol., 1989 9:875-84.; Fuller, M T et al., Cell Mot. Cyto. (1989) 14 :128-35; Rottgen G, Wagner T, Hinz U Mol. Gen. Genet. 1998 257:442-51).
Genes that mutate to enhance the adult onset neurodegeneration phenotype in a recessive manner yield potential protein therapeutics for adult onset neurodegenerative conditions, since elevating the normal gene product level of such genes potentially alleviates the neurodegenerative condition. Genes that mutate to suppress the adult onset neurodegeneration phenotype in a recessive manner yield gene targets for disruption to alleviate the neurodegenerative conditions, where disruption of these genes can be achieved using a variety of methods, ranging from deleting the DNA for the target gene to inhibiting its transcription, translation, or protein activity. For screening candidate agents, small molecule antagonists to these genes can be constructed and evaluated for efficacy in the fly model through oral administration. Alternatively, large molecular antagonists can be delivered by gene therapy, as described infra.
Kits
Also provided by the subject invention are kits for use in performing the subject screening methods. The subject kits include at least a plurality of transgenic flies of the subject invention, or a means for producing such a plurality of flies, e.g. a male and female transgenic fly of the subject invention, vectors carrying requisite genes, such as the transgene, a transposase gene, GAL4, etc. The flies may be housed in appropriate container(s), e.g. vials. The subject kits may also comprise a nutrient medium for the animals, e.g. drosophila medium.
Therapeutic Agents and Pharmaceutical Compositions
Also provided by the subject invention are therapeutic agents for use in treating a neurodegenerative condition, as well as pharmaceutical formulations thereof. The therapeutic agents of the subject invention are those agents identified using the screening methods described supra that show beneficial activity with respect to a neurodegenerative condition, particularly an adult onset neurodegenerative condition (or agents known to have an effect on the expression of a gene identified as modulating the phenotype of an adult onset neurodegenerative condition, where identification employs the use of the subject non-transgenic animals).
Also provided are pharmaceutical compositions of the subject therapeutic agents. In the pharmaceutical compositions or formulations of the subject invention, agents. described above are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose. derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term xe2x80x9cunit dosage form,xe2x80x9d as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Where the agent is a polypeptide, polynucleotide, analog or mimetic thereof (as identified using the mutant screen analysis protocols described supra, it may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or xe2x80x9cgene gunxe2x80x9d as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells.
Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
Kits with unit doses of the active agent, usually in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Preferred compounds and unit doses are those described herein above.
Methods of Treating Neurodegenerative Conditions
Also provided are methods of treating neurodegenerative disease conditions, particularly adult onset neurodegenerative disease conditions, using the subject active agents. In the subject methods, an effective amount of the active agent of the subject invention is administered to the host to be treated. By xe2x80x9ceffective amountxe2x80x9d is meant a dosage sufficient to produce a desired result, where the desired result is generally an amelioration or alleviation, if not complete cessation, of one or more symptoms of the neurodegenerative disease being treated. Administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal,etc. A variety of hosts are treatable according to the subject methods. Generally such hosts are xe2x80x9cmammalsxe2x80x9d or xe2x80x9cmammalian,xe2x80x9d where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.
Validation of Non-mammalian Animal Models for a Human Disease Condition
Also provided are methods of validating non-mammalian animal models, such as the animal models of the subject invention, as animal models for human disease conditions. In the subject validation methods, a compound having a activity with respect to he human disease of interest is administered to a potential non-mammalian animal model for said disease. The potential animal model may be one that has been identified from nature or produced via human intervention, e.g. genetic engineering. The only limitation on the putative non-mammalian animal model for the human disease of interest is that it have a phenotype that is analogous to the phenotype suffering from the human disease, i.e. it has a phenotype that can be correlated to at least one symptom of the disease in a human. For example, the animal models of the present application have a phenotype that is analogous to the phenotype of a human suffering from an adult onset neurodegenerative disease condition.
Following administration of the compound to the putative non-mammalian animal model, the effect that the compound has on the phenotype of the non-mammalian animal phenotype observed. Based on the observation, a determination is made as to whether the compound has the same effect on the phenotype of the non-mammalian animal model as it does on the human disease phenotype. For example, where the compound improves the impaired appetite in a human suffering from the human disease and also improves the impaired appetite of the putative non-mammalian animal model, the compound has the same effect on the phenotype of the animal model as it does on the phenotype of a human suffering from the disease.
If the compound has the same effect on the phenotype of the putative non-mammalian animal model as it does on a human suffering from the disease, the non-mammalian animal model is validated as an animal model for the human disease condition.
The following examples are offered by way of illustration and not by way of limitation.